The detection of cell death is a highly relevant biomarker for many biological processes related to various application fields including the severity/progression of diseases, the efficacy of various therapies and the drug safety evaluation. Cell death can proceed by several distinct pathways, including mainly apoptosis, necrosis and autophagy, which are characterized by a distinct set of temporal, morphological, biochemical, and genetic characteristics. For a detailed description of the various mechanisms involved in the different cell death pathways, see for example Duprez, L. et al. “Major cell death pathways at a glance”, Microbes Infect. 11 (13), 1050-1062 (2009).
Although, in a whole organism, certain types of cell death, including apoptosis, result in the controlled breakdown of the cell avoiding any intracellular release, in vitro, the different cell death pathways proceed to an end-stage called secondary necrosis, which shares many features with primary necrosis pathway, in particular the loss of cell membrane integrity and the subsequent release of the cellular content into the surrounding extracellular space. Consequently, in vitro, assays usually differentiate between viable and non viable cells by assessing membrane integrity thanks to inclusion and/or exclusion dyes (trypan blue or propidium iodide, for example) or the detection of specific intracellular compounds in the surrounding medium (lactate deshydrogenase release (LDH)). However, depending on the stimulus having induced cell death, such cell viability essays assess a late stage of the cell death processes with extrinsic contrast agents and usually require several steps (washing, harvesting, solubilization etc.) which take several hours for completion.
On the other hand, as mentioned before, specific morphological and biochemical features (often called “phenotypes”) accompany or are linked to cell death processes and are often used to define and recognize the different cell death pathways. For example, the loss of cell volume or cell shrinkage that occurs during apoptosis is a key morphological characteristic separating this physiological cell death process from an accidental one as necrosis, characterized by an initial cell swelling. Originally, the volume regulation is driven by homeostasis, which is the concept of the cell regulating within its environment. On a general point of view, a combination of various parameters and phenotypes linked to the homeostasis, such as protein concentration, ion concentration, water content, etc., can provide useful indicators about the cell viability.
It was indeed shown that loss of the cell normal regulatory capability is considered as a trigger for cell death. For example, cell volume deregulation was shown to be a relevant indicator for cell death triggering, and intracellular ionic concentration deregulation is seen as a primary indicator of cell biological processes dysfunction, as shown for example in Bortner, C. and Cidlowski, J., “The role of apoptotic volume decrease and ionic homeostasis in the activation and repression of apoptosis,” Pflug. Arch. Eur. J. Physiol. 448 (3), 313-318 (2004). However, minor variations of those parameters occur in accordance with the normal cell activity and cells have inherited regulatory mechanisms to compensate for these minor variations in order to maintain in particular appropriate balance of ions across their cell membrane. One can thus identify, for a given regulatory indicator, a variation range which lies in normal life cycle of the cell, and other ranges indicating a lack of regulation capacity of the cell, which can lead to cell death mechanisms triggering.